The present invention relates to Toxoplasma gondii nucleic acid molecules, proteins encoded by such nucleic acid molecules, antibodies raised against such proteins and methods to identify such nucleic acid molecules, proteins or antibodies. The present invention also includes compositions comprising such nucleic acid molecules, proteins and antibodies, as well as their use for inhibiting oocyst shedding by cats infected with T. gondii and for protecting animals from diseases caused by T. gondii. 
Various attempts to develop a vaccine to both the asexual systemic stage and the sexual entero-epithelial stage of the Toxoplasma life cycle have been reported over the last thirty years (Hermentin, K. and Aspock, H. (1988), Zbl. Bakt. Hyg. A, 269:423-436). These attempts can be grouped into the following categories: 1) immunization with whole killed organism, 2) immunization with selected antigens, either purified native or recombinant protein, 3) immunization with attenuated strains, and 4) immunization with irradiated organisms. Little success has been achieved with immunizations using whole killed organism (Frenkel, J. K. and Smith, D. D. (1982), Journal of Parasitology, 68:744-748). Partial success has been observed with the pure native protein P30 (Bulow, R., and Boothroyd, J. C. (1991), J. Immunol. 147:3496) and with selected fractions of parasite lysates (Lunden, A. Lovgren, K. Uggla, A., and Araujo, F. G.; (1993) Infection and Immunity, 61:2639-2643). However, attempts with purified recombinant antigens have not been successful (Lunden, A., Parmley, S. F., Bengtsson, K. L. and Araujo, F. G. (1997) Parasitology Research, 83:6-9). Studies with irradiated organisms have reported 0-90% protection and are complicated by the uncertainty of truly inactivated irradiated preparations. Effective vaccines have been produced using attenuated strains. Two such mutant strains, ts-4 (Waldeland, H., Pfefferkom, E. R., and Frenkel, J. K. (1983), Journal of Parasitology, 69:171-175) and S48 (Hartley, W. J. and Marshall, S. C. (1957), New Zealand Veterinary Journal, 5:119-124), successfully protect animals against the asexual systemic disease. These strains are delivered in the tachyzoite form and do not protect cats from oocyst shedding. Another strain, T-263 (Frenkel, J. K.; Pfefferkom, E. R.; Smith, D. D.; and Fishback, J. L. (1991), American Journal of Veterinary Research, 52:759-763) is an oocyst minus strain, but was shown to progress through most of the entero-epithelial stages in the cat intestine. Exposure to this strain induces immunity in the cat to oocyst shedding upon subsequent challenge. There remains a need for an effective vaccine for prevention of the diseases caused by infection with Toxoplasma gondii. 
The present invention relates to novel compositions and methods to inhibit Toxoplasma gondii (T. gondii) oocyst shedding by cats, thereby preventing the spread of T. gondii infection. According to the present invention there are provided isolated immunogenic T. gondii proteins and mimetopes thereof, T. gondii nucleic acid molecules, including those that encode such proteins; recombinant molecules including such nucleic acid molecules; recombinant viruses including such nucleic acid molecules; recombinant cells including such nucleic acid molecules; and antibodies that selectively bind to such immunogenic T. gondii proteins.
The present invention also includes methods to obtain and/or identify proteins, nucleic acid molecules, recombinant molecules, recombinant viruses, recombinant cells, and antibodies of the present invention. Also included are compositions comprising such proteins, nucleic acid molecules, recombinant molecules, recombinant viruses, recombinant cells, and antibodies, as well as use of such compositions to inhibit T. gondii oocyst shedding by cats infected with T. gondii, or for preventing T. gondii infection in an animal.
The present invention further includes the use of the nucleic acid molecules or proteins of the present invention as diagnostic reagents for the detection of T. gondii infection. In a preferred embodiment, the present invention includes a novel detection method and kit for detecting T. gondii oocysts in the feces of T. gondii infected cats.
One embodiment of the present invention is an isolated nucleic acid molecule encoding an immunogenic T. gondii protein that can be identified by a method that includes the steps of: a) immunoscreening a T. gondii genomic expression library or cDNA expression library with an antiserum, including an antiserum derived from intestinal secretions; and b) identifying a nucleic acid molecule in the library that expresses a protein that selectively binds to an antibody in the antiserum. Antisera to be used for screening include antiserum raised against T. gondii oocysts, antiserum raised against T. gondii bradyzoites, antiserum raised against T. gondii infected cat gut, and antiserum isolated from a cat immune to T. gondii infection. Another embodiment is an isolated immunogenic T. gondii protein that can be identified by a method that includes the steps of: a) immunoscreening a T. gondii genomic expression library or cDNA expression library with such an antiserum; and b) identifying a protein expressed by the library that selectively binds to antibodies in the antiserum. Also included are methods to identify and isolate such nucleic acid molecules and proteins.
The present application also includes an isolated nucleic acid molecule that hybridizes under stringent hybridization conditions with a gene that includes a nucleic acid sequence cited in Table 1. Also included in the present invention is an isolated nucleic acid molecule that hybridizes under stringent hybridization conditions with a gene that includes a nucleic acid molecule cited in Table 1. Preferred nucleic acid molecules encode immunogenic T. gondii proteins. More preferred nucleic acid molecules are those cited in Table 1.
The present invention also relates to recombinant molecules, recombinant viruses and recombinant cells that include an isolated nucleic acid molecule of the present invention. Also included are methods to produce such nucleic acid molecules, recombinant molecules, recombinant viruses and recombinant cells.
Another embodiment of the present invention is an isolated immunogenic protein encoded by a nucleic acid molecule that hybridizes under stringent hybridization conditions with a gene (i.e., with either the coding strand or the non-coding strand) comprising a nucleic acid sequence cited in Table 1 and/or a nucleic acid molecule cited in Table 1. Note that the nucleic acid molecule hybridizes with the non-coding strand of the gene, that is, with the complement of the coding strand of the gene. A preferred protein is an immunogenic T. gondii protein. More preferred proteins are those encoded by nucleic acid molecules cited in Table 1. Also preferred are the proteins cited in Table 1.
The present invention also relates to: mimetopes of immunogenic T. gondii proteins and isolated antibodies that selectively bind to immunogenic T. gondii proteins or mimetopes thereof. Also included are methods, including recombinant methods, to produce proteins, mimetopes and antibodies of the present invention.
Yet another embodiment of the present invention is a composition to inhibit T. gondii oocyst shedding in a cat due to infection with T. gondii. Such a composition includes one or more of the following protective compounds: an isolated immunogenic T. gondii protein encoded by a nucleic acid molecule that hybridizes under stringent hybridization conditions with a gene comprising a nucleic acid sequence cited in Table 1, and specifically with the non-coding-strand of that gene; an isolated antibody that selectively binds to said immunogenic T. gondii protein; and an isolated nucleic acid molecule that hybridizes under stringent hybridization conditions with a gene comprising a nucleic acid sequence cited in Table 1. Such a composition can also include an excipient, adjuvant or carrier. Preferred compositions comprising a nucleic acid molecule of the present invention include genetic vaccines, recombinant virus vaccines and recombinant cell vaccines. Also included in the present invention is a method to protect an animal, including a human, from disease caused by T. gondii, comprising the step of administering to the animal a composition of the present invention. Preferred animals to treat are cats in order to prevent oocyst shedding caused by T. gondii infection.
The present invention provides for isolated immunogenic T. gondii proteins, isolated T. gondii nucleic acid molecules including those encoding such T. gondii proteins, recombinant molecules comprising such nucleic acid molecules, recombinant viruses comprising such nucleic acid molecules, cells transformed with such nucleic acid molecules (i.e., recombinant cells), and antibodies that selectively bind to immunogenic T. gondii proteins. As used herein, the terms isolated immunogenic T. gondii protein and isolated nucleic acid molecule refer to an immunogenic T. gondii protein and a T. gondii nucleic acid molecule, respectively, derived from T. gondii which can be obtained from its natural source or can be produced using, for example, recombinant nucleic acid technology or chemical synthesis. Also included in the present invention is the use of these proteins, nucleic acid molecules, and antibodies as compositions to protect animals from diseases caused by T. gondii and to inhibit T. gondii oocyst shedding in cats. As used herein, a cat refers to any member of the cat family (i.e., Felidae), including domestic cats, wild cats and zoo cats. Examples of cats include, but are not limited to, domestic cats, lions, tigers, leopards, panthers, cougars, bobcats, lynx, jaguars, cheetahs, and servals. A preferred cat to protect is a domestic cat. Further included in the present invention is the use of these proteins, nucleic acid molecules and antibodies for the detection of T. gondii infection in an animal or as targets for the development of chemotherapeutic agents against parasitic infection.
Immunogenic T. gondii protein and nucleic acid molecules of the present invention have utility because they represent novel targets for anti-parasite vaccines or chemotherapeutic agents. Compositions of the present invention can also be used as reagents for the diagnosis of T. gondii infection in cats and other animals, including humans. The products and processes of the present invention are advantageous because they enable the inhibition of T. gondii oocyst shedding in cats, the definitive hosts for T. gondii (i.e., the animals in which T. gondii reproduction takes place). It is to be noted that the proteins and nucleic acid molecules of the present invention have uses beyond eliciting an immune response despite denoting proteins of the present invention as immunogenic proteins.
As described in more detail in the Examples, it was very difficult to isolate a nucleic acid molecule encoding an immunogenic T. gondii protein selectively bound by antisera directed against T. gondii intestinal stages. Such stages are preferred because they represent the sexual cycle of T. gondii, the preferred target for development of a composition to inhibit oocyst shedding. Unfortunately, however, the T. gondii sexual cycle cannot currently be reproduced in culture, and, there is not a simple method by which to produce a cDNA (i.e., complementary DNA) library containing only T. gondii nucleic acid molecules of various stages of the sexual cycle. For example, the infected cat gut is the source of many of the sexual stages of T. gondii, and, as such, material to be used in identifying T. gondii immunogenic proteins are contaminated with cat material. The present invention describes the development of new techniques to isolate and identify nucleic acid molecules encoding immunogenic T. gondii proteins. These techniques include (a) the isolation and enrichment of antisera against a variety of T. gondii life stages, several of which are only present in infected cats, at least predominantly in infected cat guts, and (b) the use of such antisera to screen cDNA and genomic expression libraries to identify nucleic acid molecules that express T. gondii proteins that selectively bind to such antisera.
One embodiment of the present invention is an isolated protein that includes an immunogenic T. gondii protein. The terms xe2x80x9cone or morexe2x80x9d and xe2x80x9cat least onexe2x80x9d can be used interchangeably herein. It is also to be noted that the terms xe2x80x9ccomprisingxe2x80x9d, xe2x80x9cincludingxe2x80x9d, and xe2x80x9chavingxe2x80x9d can be used interchangeably. According to the present invention, an isolated, or biologically pure, protein is a protein that has been removed from its natural milieu. The terms xe2x80x9cisolatedxe2x80x9d and xe2x80x9cbiologically purexe2x80x9d do not necessarily reflect the extent to which the protein has been purified. An isolated protein of the present invention can be obtained from its natural source, can be produced using recombinant DNA technology, or can be produced by chemical synthesis.xe2x80x9d
An isolated protein of the present invention, including a homolog, can be identified in a straight-forward manner by the protein""s ability to elicit an immune response against a naturally occurring T. gondii protein. Examples of T. gondii immunogenic proteins include proteins in which amino acids have been deleted (e.g., a truncated version of the protein, such as a peptide), inserted, inverted, substituted and/or derivatized (e.g., by glycosylation, phosphorylation, acetylation, myristoylation, prenylation, palmitoylation, amidation and/or addition of glycerophosphatidyl inositol) such that the homolog includes at least one epitope capable of eliciting an immune response against a T. gondii immunogenic protein, and/or of binding to an antibody directed against a T. gondii immunogenic protein. That is, when the homolog is administered to an animal as an immunogen, using techniques known to those skilled in the art, the animal will produce an immune response against at least one epitope of a T. gondii immunogenic protein. The ability of a protein to effect an immune response can be measured using techniques known to those skilled in the art. As used herein, the term xe2x80x9cepitopexe2x80x9d refers to the smallest portion of a protein or other antigen capable of selectively binding to the antigen binding site of an antibody or a T-cell receptor. It is well accepted by those skilled in the art that the minimal size of a protein epitope is about four to six amino acids. As is appreciated by those skilled in the art, an epitope can include amino acids that naturally are contiguous to each other as well as amino acids that, due to the tertiary structure of the natural protein, are in sufficiently close proximity to form an epitope. According to the present invention, an epitope includes a portion of a protein comprising at least about 4 amino acids, at least about 5 amino acids, at least about 6 amino acids, at least about 10 amino acids, at least about 15 amino acids, at least about 20 amino acids, at least about 25 amino acids, at least about 30 amino acids, at least about 35 amino acids, at least about 40 amino acids, at least about 50 amino acids, at least about 100 amino acids, at least about 150 amino acids, at least about 200 amino acids, at least about 250 amino acids, or at least about 300 amino acids.
Immunogenic T. gondii protein homologs can be the result of natural allelic variation or natural mutation. Immunogenic T. gondii protein homologs of the present invention can also be produced using techniques known in the art including, but not limited to, direct modifications to the protein or modifications to the gene encoding the protein using, for example, classic or recombinant DNA techniques to effect random or targeted mutagenesis.
As used herein, a nucleic acid molecule encoding an immunogenic T. gondii protein includes nucleic acid sequences related to a natural T. gondii gene. As used herein, a T. gondii gene includes all regions of the genome related to the gene, such as regulatory regions that control production of the immunogenic T. gondii protein encoded by the gene (for example, transcription, translation or post-translation control regions) as well as the coding region itself, and any introns or non-translated coding regions. As used herein, a gene that xe2x80x9cincludesxe2x80x9d or xe2x80x9ccomprisesxe2x80x9d a sequence may include that sequence in one contiguous array, or may include the sequence as fragmented exons. As used herein, the term xe2x80x9ccoding regionxe2x80x9d refers to a continuous linear array of nucleotides that translates into a protein. A full-length coding region is that coding region that is translated into a full-length protein, i.e., a complete protein as would be initially translated in its natural milieu, prior to any post-translational modifications.
In one embodiment, a T. gondii gene of the present invention includes at least one of the nucleic acid molecules cited in Table 1 (i.e., the cited nucleic acid molecules). The coding strands of the cited nucleic acid molecules are represented, respectively, by the nucleic acid sequences (i.e., the cited nucleic acid sequences) shown in Table 1. Also presented in Table 1 are the deduced amino acid sequences encoded by each of the cited nucleic acid molecules (i.e., the cited amino acid sequences) and the protein name designations (i.e., the cited proteins).
It should be noted that because nucleic acid sequencing technology is not entirely error-free, the nucleic acid sequences disclosed in the present invention (as well as other nucleic acid and protein sequences presented herein) represent the apparent nucleic acid sequences of the nucleic acid molecules encoding T. gondii proteins of the present invention. The nucleic acid molecules cited in Table 1 also include the complementary (i.e., apparently non-coding) strands. As used herein the terms xe2x80x9ccomplementary strandxe2x80x9d and xe2x80x9ccomplementxe2x80x9d refer to the nucleic acid sequence of the DNA strand that is fully complementary to the DNA strand having the listed sequence, which can easily be determined by those skilled in the art. Likewise, a nucleic acid sequence complement of any nucleic acid sequence of the present invention refers to the nucleic acid sequence of the nucleic acid strand that is fully complementary to (i.e., can form a complete double helix with) the strand for which the sequence is cited. Production of the cited nucleic acid molecules is disclosed in the Examples as are methods to obtain nucleic acid sequences of the coding strands of such molecules and the amino acid sequences deduced therefrom.
In another embodiment, a T. gondii gene or nucleic acid molecule can be a naturally occurring allelic variant that includes a similar but not identical sequence to the cited nucleic acid molecules. A naturally occurring allelic variant of a T. gondii gene including any of the above-listed nucleic acid sequences is a gene that occurs at essentially the same locus (or loci) in the genome as the gene including at least one of the above-listed sequences, but which, due to natural variations caused by, for example, mutation or recombination, has a similar but not identical sequence. Because natural selection typically selects against alterations that affect function, allelic variants usually encode proteins having similar activity to that of the protein encoded by the gene to which they are being compared. Allelic variants of genes or nucleic acid molecules can also comprise alterations in the 5xe2x80x2 or 3xe2x80x2 untranslated regions of the gene (e.g., in regulatory control regions), or can involve alternative splicing of a nascent transcript, thereby bringing alternative exons into juxtaposition. Allelic variants are well known to those skilled in the art and would be expected to be found within a given T. gondii organism or population, because, for example, the genome goes through a diploid stage, and sexual reproduction results in the reassortment of alleles.
In one embodiment of the present invention, an isolated immunogenic T. gondii protein is encoded by a nucleic acid molecule that hybridizes under stringent hybridization conditions to a gene encoding an immunogenic T. gondii protein. The minimal size of a T. gondii protein of the present invention is a size sufficient to be encoded by a nucleic acid molecule capable of forming a stable hybrid (i.e., hybridizing under stringent hybridization conditions) with the complementary sequence of a nucleic acid molecule encoding the corresponding natural protein. The size of a nucleic acid molecule encoding such a protein is dependent on the nucleic acid composition and the percent homology between the T. gondii nucleic acid molecule and the complementary nucleic acid sequence. It can easily be understood that the extent of homology required to form a stable hybrid under stringent conditions can vary depending on whether the homologous sequences are interspersed throughout a given nucleic acid molecule or are clustered (i.e., localized) in distinct regions on a given nucleic acid molecule.
The minimal size of a nucleic acid molecule capable of forming a stable hybrid with a gene encoding an immunogenic T. gondii protein is typically at least about 12 to about 15 nucleotides in length if the nucleic acid molecule is GC-rich and at least about 15 to about 17 bases in length if it is AT-rich. The minimal size of a nucleic acid molecule used to encode an immunogenic T. gondii protein homolog of the present invention is from about 12 to about 18 nucleotides in length. Thus, the minimal size of an immunogenic T. gondii protein homolog of the present invention is from about 4 to about 6 amino acids in length. There is no limit, other than a practical limit, on the maximal size of a nucleic acid molecule encoding an immunogenic T. gondii protein of the present invention because a nucleic acid molecule of the present invention can include a portion of a gene, an entire gene, or multiple genes. A preferred nucleic acid molecule of the present invention is a nucleic acid molecule that is at least 12 nucleotides in length. Also preferred are nucleic acid molecules that are at least 18 nucleotides, or at least 20 nucleotides, or at least 25 nucleotides, or at least 30 nucleotides, or at least 40 nucleotides, or at least 50 nucleotides, or at least 70 nucleotides, or at least 100 nucleotides, or at least 150 nucleotides, or at least 200 nucleotides, or at least 250 nucleotides, or at least 300 nucleotides, or at least 350 nucleotides, or at least 400 nucleotides, or at least 500 nucleotides, or at least 750 nucleotides, or at least 1000 nucleotides, or at least 1500 nucleotides, or at least 1750 nucleotides, or at least 2000 nucleotides, or at least 2250 nucleotides, or at least 2417 nucleotides in length, The preferred size of a protein encoded by a nucleic acid molecule of the present invention depends on whether a full-length, fusion, multivalent, or functional portion of such a protein is desired.
Stringent hybridization conditions are determined based on defined physical properties of the gene to which the nucleic acid molecule is being hybridized, and can be defined mathematically. Stringent hybridization conditions are those experimental parameters that allow an individual skilled in the art to identify significant similarities between heterologous nucleic acid molecules. These conditions are well known to those skilled in the art. See, for example, Sambrook, et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Labs Press, and Meinkoth, et al., 1984, Anal. Biochem. 138, 267-284, each of which is incorporated by reference herein in its entirety. As explained in detail in the cited references, the determination of hybridization conditions involves the manipulation of a set of variables including the ionic strength (M, in moles/liter), the hybridization temperature (xc2x0C.), the concentration of nucleic acid helix destabilizing agents (such as formamide), the average length of the shortest hybrid duplex (n), and the percent G+C composition of the fragment to which an unknown nucleic acid molecule is being hybridized. For nucleic acid molecules of at least about 150 nucleotides, these variables are inserted into a standard mathematical formula to calculate the melting temperature, or Tm, of a given nucleic acid molecule. As defined in the formula below, Tm is the temperature at which two complementary nucleic acid molecule strands will disassociate, assuming 100% complementarity between the two strands:
Tm=81.5xc2x0 C.+16.6 log M+0.41(% G+C)xe2x88x92500/nxe2x88x920.61(% formamide). 
For nucleic acid molecules smaller than about 50 nucleotides, hybrid stability is defined by the dissociation temperature (Td), which is defined as the temperature at which 50% of the duplexes dissociate. For these smaller molecules, the stability at a standard ionic strength is defined by the following equation:
Td=4(G+C)+2(A+T). 
A temperature of 5xc2x0 C. below Td is used to detect hybridization between perfectly matched molecules.
Also well known to those skilled in the art is how base-pair mismatch, i.e. differences between two nucleic acid molecules being compared, including non-complementarity of bases at a given location, and gaps due to insertion or deletion of one or more bases at a given location on either of the nucleic acid molecules being compared, will affect Tm or Td for nucleic acid molecules of different sizes. For example, Tm decreases about 1xc2x0 C. for each 1% of mismatched base-pairs for hybrids greater than about 150 bp, and Td decreases about 5xc2x0 C. for each mismatched base-pair for hybrids below about 50 bp. Conditions for hybrids between about 50 and about 150 base-pairs can be determined empirically and without undue experimentation using standard laboratory procedures well known to those skilled in the art. These simple procedures allow one skilled in the art to set the hybridization conditions (by altering, for example, the salt concentration, the formamide concentration or the temperature) so that only nucleic acid hybrids with less than a specified % base-pair mismatch will hybridize. Stringent hybridization conditions are commonly understood by those skilled in the art to be those experimental conditions that will allow hybridization between molecules having about 30% or less base-pair mismatch (i.e., about 70% or greater identity). Because one skilled in the art can easily determine whether a given nucleic acid molecule to be tested is less than or greater than about 50 nucleotides, and can therefore choose the appropriate formula for determining hybridization conditions, he or she can determine whether the nucleic acid molecule will hybridize with a given gene under stringent hybridization conditions and similarly whether the nucleic acid molecule will hybridize under conditions designed to allow a desired amount of base pair mismatch.
Hybridization reactions are often carried out by attaching the nucleic acid molecule to be hybridized to a solid support such as a membrane, and then hybridizing with a labeled nucleic acid molecule, typically referred to as a probe, suspended in a hybridization solution. Examples of common hybridization reaction techniques include, but are not limited to, the well-known Southern and northern blotting procedures. Typically, the actual hybridization reaction is done under non-stringent conditions, i.e., at a lower temperature and/or a higher salt concentration, and then high stringency is achieved by washing the membrane in a solution with a higher temperature and/or lower salt concentration in order to achieve the desired stringency.
For example, if the skilled artisan wished to identify a nucleic acid molecule that hybridizes under stringent hybridization conditions with a T. gondii nucleic acid molecule of about 150 bp in length, the following conditions could preferably be used. As an example, the average G+C content of Dirofilaria immitis DNA is about 35%. The unknown nucleic acid molecules would be attached to a support membrane, and the 150 bp probe would be labeled, e.g. with a radioactive tag. The hybridization reaction could be carried out in a solution comprising 2xc3x97SSC and 0% formamide, at a temperature of about 37xc2x0 C. (low stringency conditions). Solutions of differing concentrations of SSC can be made by one of skill in the art by diluting a stock solution of 20xc3x97SSC (175.3 gram NaCl and about 88.2 gram sodium citrate in 1 liter of water, pH 7) to obtain the desired concentration of SSC. In order to achieve high stringency hybridization, the skilled artisan would calculate the washing conditions required to allow up to 30% base-pair mismatch. For example, in a wash solution comprising 1xc3x97SSC and 0% formamide, the Tm of perfect hybrids would be about 79xc2x0 C.:
81.5xc2x0 C.+16.6 log (0.15M)+(0.41xc3x9735)xe2x88x92(500/150)xe2x88x92(0.61xc3x970)=79xc2x0 C. 
Thus, to achieve hybridization with nucleic acid molecules having about 30% base-pair mismatch, hybridization washes would be carried out at a temperature of about 49xc2x0 C. It is thus within the skill of one in the art to calculate additional hybridization temperatures based on the desired percentage base-pair mismatch, formulae and G/C content disclosed herein. For example, it is appreciated by one skilled in the art that as the nucleic acid molecule to be tested for hybridization against nucleic acid molecules of the present invention having sequences specified herein becomes longer than 150 nucleotides, the Tm for a hybridization reaction allowing up to 30% base-pair mismatch will not vary significantly from 49xc2x0 C.
Furthermore, it is known in the art that there are commercially available computer programs for determining the degree of similarity between two nucleic acid sequences. These computer programs include various known methods to determine the percentage identity and the number and length of gaps between hybrid nucleic acid molecules. Preferred methods to determine the percent identity among amino acid sequences and also among nucleic acid sequences include analysis using one or more of the commercially available computer programs designed to compare and analyze nucleic acid or amino acid sequences. These computer programs include, but are not limited to, GCG(trademark) (available from Genetics Computer Group, Madison, Wis.), DNAsis(trademark) (available from Hitachi Software, San Bruno, Calif.) and MacVector(trademark) (available from the Eastman Kodak Company, New Haven, Conn.). A preferred method to determine percent identity among amino acid sequences and also among nucleic acid sequences includes using the GCG(trademark) program, Bestfit function with default parameter settings, or a gap weight of 12, a length weight of 4, an average match of 2.912, and an average mismatch of xe2x88x922.003.
A preferred immunogenic T. gondii protein of the present invention is a compound that, when administered to an animal in an effective manner, is capable of protecting that animal from disease caused by T. gondii or, in the case of cats, is capable of preventing T. gondii oocyst shedding in cats infected with T. gondii. In accordance with the present invention, the ability of an immunogenic T. gondii protein of the present invention to protect an animal from T. gondii disease refers to the ability of that protein to, for example, treat, ameliorate and/or prevent disease caused by T. gondii. In one embodiment, an immunogenic T. gondii protein of the present invention can elicit an immune response (including a humoral and/or cellular immune response) against T. gondii. 
The present invention also includes mimetopes of immunogenic T. gondii proteins of the present invention. As used herein, a mimetope of an immunogenic T. gondii protein of the present invention refers to any compound that is able to mimic the activity of such an immunogenic T. gondii protein, often because the mimetope has a structure that mimics the particular T. gondii protein. Mimetopes can be, but are not limited to: peptides that have been modified to decrease their susceptibility to degradation such as all-D retro peptides; anti-idiotypic and/or catalytic antibodies, or fragments thereof; non-proteinaceous immunogenic portions of an isolated protein (e.g., carbohydrate structures); and synthetic or natural organic molecules, including nucleic acids. Such mimetopes can be designed using computer-generated structures of proteins of the present invention. Mimetopes can also be obtained by generating random samples of molecules, such as oligonucleotides, peptides or other organic molecules, and screening such samples by affinity chromatography techniques using the corresponding binding partner.
One embodiment of an immunogenic T. gondii protein of the present invention is a fusion protein that includes an immunogenic T. gondii protein-containing domain attached to one or more fusion segments. Suitable fusion segments for use with the present invention include, but are not limited to, segments that can: enhance a protein""s stability; act as an immunopotentiator to enhance an immune response against an immunogenic T. gondii protein; and/or assist in purification of an immunogenic T. gondii protein (e.g., by affinity chromatography). A suitable fusion segment can be a domain of any size that has the desired function (e.g., imparts increased stability, imparts increased immunogenicity to a protein, and/or simplifies purification of a protein). Fusion segments can be joined to amino and/or carboxyl termini of the immunogenic T. gondii protein-containing domain of the protein and can be susceptible to cleavage in order to enable straightforward recovery of an immunogenic T. gondii protein. Fusion proteins are preferably produced by culturing a recombinant cell transformed with a nucleic acid molecule that encodes a protein including the fusion segment attached to either the carboxyl and/or amino terminal end of an immunogenic T. gondii protein-containing domain. Preferred fusion segments include a metal binding domain (e.g., a poly-histidine segment); an immunoglobulin binding domain (e.g., Protein A; Protein G; T cell; B cell; Fc receptor or complement protein antibody-binding domains); a sugar binding domain (e.g., a maltose binding domain); and/or a xe2x80x9ctagxe2x80x9d domain (e.g., at least a portion of xcex2-galactosidase, a strep tag peptide, a T7 tag peptide, a Flag(trademark) peptide, or other domains that can be purified using compounds that bind to the domain, such as monoclonal antibodies). More preferred fusion segments include metal binding domains, such as a poly-histidine segment; a maltose binding domain; a strep tag peptide, such as that available from Biometra in Tampa, Fla.; and an S10 peptide.
In another embodiment, an immunogenic T. gondii protein of the present invention also includes at least one additional protein segment that is capable of protecting an animal from one or more diseases. Such a multivalent protective protein can be produced, for example, by culturing a cell transformed with a nucleic acid molecule comprising two or more nucleic acid domains joined together in such a manner that the resulting nucleic acid molecule is expressed as a multivalent protective compound containing at least two protective compounds capable of protecting an animal from diseases caused, for example, by at least one infectious agent.
Examples of multivalent protective compounds include, but are not limited to, an immunogenic T. gondii protein of the present invention attached to one or more compounds protective against one or more other infectious agents, particularly an agent that infects cats. In another embodiment, one or more protective compounds can be included in a multivalent vaccine comprising an immunogenic T. gondii protein of the present invention and one or more other protective molecules as separate compounds.
A preferred isolated immunogenic T. gondii protein of the present invention includes a protein that is encoded by a nucleic acid molecule that hybridizes under stringent hybridization conditions with a gene (i.e., with the non-coding strand which is a complement of the coding strand) comprising at least one of the nucleic acid molecules cited in Table 1. As such, also preferred is a protein that is encoded by a nucleic acid molecule that hybridizes under stringent hybridization conditions with the non-coding strand of a gene comprising at least one of the nucleic acid sequences cited in Table 1. More preferred is a protein encoded by a nucleic acid molecule that hybridizes under stringent hybridization conditions with at least one of the cited nucleic acid molecules particularly since those nucleic acid molecules have been shown to encode proteins that selectively bind to antiserum that either was raised against T. gondii oocysts, bradyzoites, or infected cat gut, or was isolated from a cat immune to T. gondii infection. As such, also preferred is a protein encoded by a nucleic acid molecule that hybridizes under stringent hybridization conditions with the complement of at least one of the cited nucleic acid sequences.
Even more preferred are isolated proteins having an amino acid sequence encoded by a nucleic acid molecules that are at least about 75%, preferably at least about 80%, more preferably at least about 85%, even more preferably at least about 90%, even more preferably at least about 95%, and even more preferably at least about 98% identical to one of the nucleic acid molecules and/or nucleic acid sequences cited in Table 1. Also preferred are proteins that comprise one or more epitopes of any of the proteins having such amino acid sequences.
A particularly preferred isolated protein of the present invention is a protein having an amino acid sequence encoded by at least one of the cited nucleic acid molecules and/or cited nucleic acid sequences, a protein encoded by an allelic variant of at least one of the cited nucleic acid molecules and/or nucleic acid sequences, or a protein comprising an epitope of any of the proteins having such amino acid sequences.
In one embodiment, preferred immunogenic T. gondii proteins of the present invention include proteins that are at least about 75%, preferably at least about 80%, more preferably at least about 85%, even more preferably at least about 90%, and even more preferably at least about 95% identical to at least one of the proteins cited in Table 1. As such, also preferred are proteins that are at least about 75%, preferably at least about 80%, more preferably at least about 85%, even more preferably at least about 90%, and even more preferably at least about 95% identical to at least one of the amino acid sequences cited in Table 1. Also preferred are proteins that comprise one or more epitopes of any of such proteins. More preferred are immunogenic T. gondii proteins comprising the cited proteins and/or having the cited amino acid sequences, proteins encoded by allelic variants of nucleic acid molecules encoding proteins including the cited proteins and/or having the cited amino acid sequences, and proteins having one or more epitopes of such proteins.
Another embodiment of the present invention is an isolated nucleic acid molecule comprising a T. gondii nucleic acid molecule that encodes an immunogenic T. gondii protein. The identifying characteristics of such nucleic acid molecules are heretofore described. A nucleic acid molecule of the present invention can include an isolated natural T. gondii nucleic acid molecule or a homolog thereof, the latter of which is described in more detail below. A nucleic acid molecule of the present invention can include one or more regulatory regions, full-length or partial coding regions, or combinations thereof. The minimal size of a nucleic acid molecule of the present invention is a size sufficient to allow the formation of a stable hybrid (i.e., hybridization under stringent hybridization conditions) with the complementary sequence of another nucleic acid molecule. The minimal size of an T. gondii nucleic acid molecule of the present invention is from about 12 to about 18 nucleotides in length.
In accordance with the present invention, an isolated nucleic acid molecule is a nucleic acid molecule that has been removed from its natural milieu (i.e., that has been subjected to human manipulation) and can include DNA, RNA, or derivatives of either DNA or RNA. Accordingly, the term xe2x80x9cisolatedxe2x80x9d, as used herein to describe a nucleic acid molecule, does not reflect the extent to which the nucleic acid molecule has been purified. An isolated T. gondii nucleic acid molecule of the present invention can be isolated from its natural source or produced using recombinant nucleic acid technology (e.g., polymerase chain reaction (PCR) amplification or cloning) or chemical synthesis. Isolated T. gondii nucleic acid molecules can include, for example, natural allelic variants and nucleic acid molecules modified by nucleotide insertions, deletions, substitutions, and/or inversions in a manner such that the modifications do not substantially interfere with the nucleic acid molecule""s ability to encode an immunogenic T. gondii protein of the present invention.
A homolog of a nucleic acid molecule encoding an immunogenic T. gondii protein can be produced using a number of methods known to those skilled in the art, see, for example, Sambrook et al., 1989, Molecular Cloning. A Laboratory Manual, Cold Spring Harbor Labs Press; Sambrook et al., ibid., is incorporated by reference herein in its entirety. For example, nucleic acid molecules can be modified using a variety of techniques including, but not limited to, classic mutagenesis and recombinant DNA techniques such as site-directed mutagenesis, chemical treatment, restriction enzyme cleavage, ligation of nucleic acid fragments, PCR amplification, synthesis of oligonucleotide mixtures and ligation of mixture groups to xe2x80x9cbuildxe2x80x9d a mixture of nucleic acid molecules, and combinations thereof. Nucleic acid molecule homologs can be selected by hybridization with a nucleic acid molecule encoding an immunogenic T. gondii protein or by screening the function of a protein encoded by the nucleic acid molecule (e.g., ability to elicit an immune response against at least one epitope of an immunogenic T. gondii protein).
An isolated nucleic acid molecule of the present invention can include a nucleic acid sequence that encodes at least one immunogenic T. gondii protein of the present invention, examples of which are disclosed herein. Although the phrase xe2x80x9cnucleic acid moleculexe2x80x9d primarily refers to the physical nucleic acid molecule and the phrase xe2x80x9cnucleic acid sequencexe2x80x9d primarily refers to the sequence of nucleotides on the nucleic acid molecule, the two phrases can be used interchangeably, especially with respect to a nucleic acid molecule, or a nucleic acid sequence, capable of encoding an T. gondii protein.
A preferred nucleic acid molecule of the present invention, when administered to a cat, is capable of preventing T. gondii oocyst shedding. As will be disclosed in more detail below, such a nucleic acid molecule can be, or encode, an antisense RNA, a molecule capable of triple helix formation, a ribozyme, or other nucleic acid-based drug compound. In additional embodiments, a nucleic acid molecule of the present invention can encode a protective protein (e.g., an immunogenic T. gondii protein of the present invention), the nucleic acid molecule being delivered to the animal, for example, by direct injection (i.e, as a genetic vaccine) or in a vehicle such as a recombinant virus vaccine or a recombinant cell vaccine. Another preferred nucleic acid molecule of the present invention, when administered to an animal, is capable of preventing disease in that animal caused by T. gondii. 
One embodiment of the present invention is an isolated nucleic acid molecule that hybridizes under stringent hybridization conditions with a nucleic acid molecule comprising at least one of the nucleic acid molecules cited in Table 1. As such, also preferred is a nucleic acid molecule that hybridizes under stringent hybridization conditions with at least one of the nucleic acid sequences cited in Table 1 or with a complement of such a sequence. More preferred is a nucleic acid molecule that hybridizes under stringent hybridization conditions with at least one of the cited nucleic acid molecules. As such, also preferred is a nucleic acid molecule that hybridizes under stringent hybridization conditions with at least one of the cited nucleic acid sequences or with a complement thereof.
Even more preferred are isolated nucleic acid molecules that are at least about 75%, preferably at least about 80%, more preferably at least about 85%, even more preferably at least about 90%, even more preferably at least about 95%, and even more preferably at least about 98% identical to one of the nucleic acid molecules and/or nucleic acid sequences cited in Table 1. Also preferred are nucleic acid molecules that form stable hybrids with nucleic acid molecules having those percent identities.
A particularly preferred isolated nucleic acid molecule of the present invention is a nucleic acid molecule that comprises at least one of the cited nucleic acid molecules and/or cited nucleic acid sequences, a nucleic acid molecule that is an allelic variant of at least one of the cited nucleic acid molecules and/or nucleic acid sequences, or a nucleic acid molecule that is a portion thereof (i.e., a nucleic acid molecule that forms a stable hybrid with at least one of the cited nucleic acid molecules or allelic variants thereof).
In one embodiment, a nucleic acid molecule encoding an immunogenic T. gondii protein of the present invention encodes a protein that is at least about 75%, preferably at least about 80%, more preferably at least about 85%, even more preferably at least about 90%, and even more preferably at least about 95% identical to the proteins cited in Table 1. Even more preferred is a nucleic acid molecule encoding a protein cited in Table 1 or an allelic variant of such a nucleic acid molecule. Also preferred are nucleic acid molecules encoding proteins comprising one or more epitopes of proteins having the cited percent identities or epitopes of proteins cited in Table 1 or encoded by nucleic acid molecules that are allelic variants of nucleic acid molecules cited in Table 1.
In another embodiment, a nucleic acid molecule encoding an immunogenic T. gondii protein of the present invention encodes a protein having an amino acid sequence that is at least about 75%, preferably at least about 80%, more preferably at least about 85%, even more preferably at least about 90%, and even more preferably at least about 95% identical to at least one of the amino acid sequences cited in Table 1. Even more preferred is a nucleic acid molecule encoding a protein having an amino acid sequence cited in Table 1 or an allelic variant of such a nucleic acid molecule. Also preferred are nucleic acid molecules encoding proteins comprising one or more epitopes of proteins having the cited percent identities or epitopes of proteins having amino acid sequences cited in Table 1 or encoded by nucleic acid molecules that are allelic variants of nucleic acid molecules cited in Table 1.
Note that nucleic acid molecules of the present invention can include nucleotide sequences in addition to those disclosed above, such as, but not limited to, nucleotide sequences comprising a full-length gene, a full-length coding region, a nucleic acid molecule encoding a fusion protein, or a nucleic acid molecule encoding a multivalent protective compound. Also included in the present invention are nucleic acid molecules that have been modified to accommodate codon usage properties of the cells in which such nucleic acid molecules are to be expressed. Preferred nucleic acid molecules of the present invention include fragments of the nucleic acid molecules disclosed in Table 1.
Knowing the nucleic acid sequences of certain nucleic acid molecules encoding immunogenic T. gondii proteins of the present invention allows one skilled in the art to, for example, (a) make copies of those nucleic acid molecules, (b) obtain nucleic acid molecules including at least a portion of such nucleic acid molecules (e.g., nucleic acid molecules including full-length genes, full-length coding regions, regulatory control sequences, truncated coding regions), and (c) obtain other nucleic acid molecules encoding an immunogenic T. gondii proteins. Such nucleic acid molecules can be obtained in a variety of ways including screening appropriate expression libraries with antibodies of the present invention; traditional cloning techniques using oligonucleotide probes of the present invention to screen appropriate libraries; and PCR amplification of appropriate libraries or DNA using oligonucleotide primers of the present invention. Preferred libraries to screen or from which to amplify nucleic acid molecules include T. gondii cDNA libraries as well as genomic DNA libraries. Similarly, preferred DNA sources from which to amplify nucleic acid molecules include T. gondii EDNA and genomic DNA. Techniques to clone and amplify nucleic acid molecules are disclosed, for example, in Sambrook et al., ibid.
The present invention also includes nucleic acid molecules that are oligonucleotides capable of hybridizing, under stringent hybridization conditions, with complementary regions of other, preferably longer, nucleic acid molecules of the present invention such as those comprising nucleic acid molecules encoding immunogenic T. gondii proteins. Oligonucleotides of the present invention can be RNA, DNA, or derivatives of either. The minimum size of such oligonucleotides is the size required for formation of a stable hybrid between an oligonucleotide and a complementary sequence on a nucleic acid molecule of the present invention. A preferred oligonucleotide of the present invention has a maximum size of about 100 nucleotides. The present invention includes oligonucleotides that can be used as, for example, probes to identify nucleic acid molecules encoding immunogenic T. gondii proteins, primers to produce nucleic acid molecules encoding immunogenic T. gondii proteins, or reagents to inhibit immunogenic T. gondii protein production or activity (e.g., as antisense-, triplex formation-, ribozyme- and/or RNA drug-based reagents). The present invention also includes the use of such oligonucleotides to protect animals from disease using one or more of such technologies. Appropriate oligonucleotide-containing compositions can be administered to an animal using techniques known to those skilled in the art.
One embodiment of the present invention includes a recombinant vector, which includes at least one isolated nucleic acid molecule of the present invention, inserted into any vector capable of delivering the nucleic acid molecule into a host cell. Such a vector contains heterologous nucleic acid sequences, that is nucleic acid sequences that are not naturally found adjacent to nucleic acid molecules of the present invention and that preferably are derived from a species other than the species from which the nucleic acid molecule(s) are derived. The vector can be either RNA or DNA, either prokaryotic or eukaryotic, and typically is a virus or a plasmid. Recombinant vectors can be used in the cloning, sequencing, and/or otherwise manipulating of nucleic acid molecule encoding immunogenic T. gondii proteins of the present invention.
One type of recombinant vector, referred to herein as a recombinant molecule, comprises a nucleic acid molecule of the present invention operatively linked to an expression vector. The phrase operatively linked refers to insertion of a nucleic acid molecule into an expression vector in a manner such that the molecule is able to be expressed when transformed into a host cell. As used herein, an expression vector is a DNA or RNA vector that is capable of transforming a host cell and of effecting expression of a specified nucleic acid molecule. Preferably, the expression vector is also capable of replicating within the host cell. Expression vectors can be operative in either prokaryotic or eukaryotic cells, and are typically viruses or plasmids. Expression vectors of the present invention include any vectors that function (i.e., direct gene expression) in recombinant cells of the present invention, including in bacterial, fungal, parasite, insect, other animal, and plant cells. Preferred expression vectors of the present invention can direct gene expression in bacterial, yeast, T. gondii and mammalian cells, and more preferably in the cell types disclosed herein.
In particular, expression vectors of the present invention contain regulatory sequences such as transcription control sequences, translation control sequences, origins of replication, and other regulatory sequences that are compatible with the recombinant cell and that control the expression of nucleic acid molecules of the present invention. In particular, recombinant molecules of the present invention include transcription control sequences. Transcription control sequences are sequences which control the initiation, elongation, and termination of transcription. Particularly important transcription control sequences are those which control transcription initiation, such as promoter, enhancer, operator and repressor sequences. Suitable transcription control sequences include any transcription control sequence that can function in at least one of the recombinant cells of the present invention. A variety of such transcription control sequences are known to those skilled in the art. Preferred transcription control sequences include those which function in bacterial, yeast, helminth or other endoparasite, or insect and mammalian cells, such as, but not limited to, tac, lac, trp, trc, oxy-pro, omp/lpp, rrnB, bacteriophage lambda (such as lambda pL and lambda pR and fusions that include such promoters), bacteriophage T7, T7lac, bacteriophage T3, bacteriophage SP6, bacteriophage SP01, metallothionein, alpha-mating factor, Pichia alcohol oxidase, alphavirus subgenomic promoter, antibiotic resistance gene, baculovirus, Heliothis zea insect virus, vaccinia virus, herpesvirus, raccoon poxvirus, other poxvirus, adenovirus, cytomegalovirus (such as immediate early promoter), simian virus 40, retrovirus, actin, retroviral long terminal repeat, Rous sarcoma virus, heat shock, phosphate and nitrate transcription control sequences as well as other sequences capable of controlling gene expression in prokaryotic or eukaryotic cells. Additional suitable transcription control sequences include tissue-specific promoters and enhancers as well as lymphokine-inducible promoters (e.g., promoters inducible by interferons or interleukins). Transcription control sequences of the present invention can also include naturally occurring transcription control sequences naturally associated with T. gondii. 
Suitable and preferred nucleic acid molecules to include in recombinant vectors of the present invention are as disclosed herein. Preferred nucleic acid molecules to include in recombinant vectors, and particularly in recombinant molecules, include those cited in Table 1. Particularly preferred recombinant molecules of the present invention include those recombinant molecules, the production of which are described in the Examples section.
Recombinant molecules of the present invention may also (a) contain secretory signals (i.e., signal segment nucleic acid sequences) to enable an expressed T. gondii protein of the present invention to be secreted from the cell that produces the protein and/or (b) contain fusion sequences which lead to the expression of nucleic acid molecules of the present invention as fusion proteins. Examples of suitable signal segments include any signal segment capable of directing the secretion of a protein of the present invention. Preferred signal segments include, but are not limited to, tissue plasminogen activator (t-PA), interferon, interleukin, growth hormone, histocompatibility and viral envelope glycoprotein signal segments. Suitable fusion segments encoded by fusion segment nucleic acids are disclosed herein. In addition, a nucleic acid molecule of the present invention can be joined to a fusion segment that directs the encoded protein to the proteosome, such as a ubiquitin fusion segment. Eukaryotic recombinant molecules may also include intervening and/or untranslated sequences surrounding and/or within the nucleic acid sequences of nucleic acid molecules of the present invention.
Another embodiment of the present invention includes a recombinant cell comprising a host cell transformed with one or more recombinant molecules of the present invention. Transformation of a nucleic acid molecule into a cell can be accomplished by any method by which a nucleic acid molecule can be inserted into the cell. Transformation techniques include, but are not limited to, transfection, electroporation, microinjection, lipofection, adsorption, and protoplast fusion. A recombinant cell may remain unicellular or may grow into a tissue, organ or a multicellular organism. Transformed nucleic acid molecules of the present invention can remain extrachromosomal or can integrate into one or more sites within a chromosome of the transformed (i.e., recombinant) cell in such a manner that their ability to be expressed is retained. Preferred nucleic acid molecules with which to transform a cell include nucleic acid molecules encoding immunogenic T. gondii proteins disclosed herein. Particularly preferred nucleic acid molecules with which to transform a cell include those listed in Table 1.
Suitable host cells to transform include any cell that can be transformed with a nucleic acid molecule of the present invention. Host cells can be either untransformed cells or cells that are already transformed with at least one nucleic acid molecule (e.g., nucleic acid molecules encoding one or more proteins of the present invention and/or other proteins useful in the production of multivalent vaccines). Host cells of the present invention either can be endogenously (i.e., naturally) capable of producing T. gondii proteins of the present invention or can be capable of producing such proteins after being transformed with at least one nucleic acid molecule of the present invention. Host cells of the present invention can be any cell capable of producing at least one protein of the present invention, and include bacterial, fungal (including yeast), parasite (including helminth, protozoa and ectoparasite), insect, other animal and plant cells. Preferred host cells include bacterial, mycobacterial, yeast, protozoan, helminth, insect and mammalian cells. More preferred host cells include Salmonella, Escherichia, Bacillus, Listeria, Saccharomyces, Spodoptera, Mycobacteria, Trichoplusia, BHK (baby hamster kidney) cells, MDCK cells (Madin-Darby canine kidney cell line), CRFK cells (Crandell feline kidney cell line), CV-1 cells (African monkey kidney cell line used, for example, to culture raccoon poxvirus), COS (e.g., COS-7) cells, and Vero cells. Particularly preferred host cells are Escherichia coli, including E. coli K-12 derivatives; Salmonella typhi; Salmonella typhimurium, including attenuated strains such as UK-1 "khgr"3987 and SR-11 "khgr"4072; Spodoptera frugiperda; Trichoplusia ni; BHK cells; MDCK cells; CRFK cells; CV-1 cells; COS cells; Vero cells; and non-tumorigenic mouse myoblast G8 cells (e.g., ATCC CRL 1246). Additional appropriate mammalian cell hosts include other kidney cell lines, other fibroblast cell lines (e.g., human, murine or chicken embryo fibroblast cell lines), myeloma cell lines, Chinese hamster ovary cells, mouse NIH/3T3 cells, LMTK31 cells and/or HeLa cells. In one embodiment, the proteins may be expressed as heterologous proteins in myeloma cell lines employing immunoglobulin promoters.
A recombinant cell is preferably produced by transforming a host cell with one or more recombinant molecules, each comprising one or more nucleic acid molecules of the present invention operatively linked to an expression vector containing one or more transcription control sequences, examples of which are disclosed herein.
A recombinant cell of the present invention includes any cell transformed with at least one of any nucleic acid molecule of the present invention. Suitable and preferred nucleic acid molecules as well as suitable and preferred recombinant molecules with which to transfer cells are disclosed herein. Particularly preferred recombinant cells include those recombinant cells, the production of which are disclosed in the Examples section.
Recombinant cells of the present invention can also be co-transformed with one or more recombinant molecules including a nucleic acid molecule encoding at least one immunogenic T. gondii protein of the present invention and one or more other nucleic acid molecules encoding other protective compounds, as disclosed herein (e.g., to produce multivalent vaccines).
Recombinant DNA technologies can be used to improve expression of transformed nucleic acid molecules by manipulating, for example, the number of copies of the nucleic acid molecules within a host cell, the efficiency with which those nucleic acid molecules are transcribed, the efficiency with which the resultant transcripts are translated, and the efficiency of post-translational modifications. Recombinant techniques useful for increasing the expression of nucleic acid molecules of the present invention include, but are not limited to, operatively linking nucleic acid molecules to high-copy number plasmids, integration of the nucleic acid molecules into one or more host cell chromosomes, addition of vector stability sequences to plasmids, substitutions or modifications of transcription control signals (e.g., promoters, operators, enhancers), substitutions or modifications of translational control signals (e.g., ribosome binding sites, Shine-Dalgarno sequences), modification of nucleic acid molecules of the present invention to correspond to the codon usage of the host cell, deletion of sequences that destabilize transcripts, and use of control signals that temporally separate recombinant cell growth from recombinant enzyme production during fermentation. The activity of an expressed recombinant protein of the present invention may be improved by fragmenting, modifying, or derivatizing nucleic acid molecules encoding such a protein.
Isolated T. gondii proteins of the present invention can be produced in a variety of ways, including production and recovery of natural proteins, production and recovery of recombinant proteins, and chemical synthesis of the proteins. In one embodiment, an isolated protein of the present invention is produced by culturing a cell capable of expressing the protein under conditions effective to produce the protein, and recovering the protein. A preferred cell to culture is a recombinant cell of the present invention. Effective culture conditions include, but are not limited to, effective media, bioreactor, temperature, pH and oxygen conditions that permit protein production. An effective medium refers to any medium in which a cell is cultured to produce an immunogenic T. gondii protein of the present invention. Effective media typically comprise an aqueous medium having assimilable carbon, nitrogen and phosphate sources, and appropriate salts, minerals, metals and other nutrients, such as vitamins. Cells of the present invention can be cultured in conventional fermentation bioreactors, shake flasks, test tubes, microtiter dishes, and petri plates. Culturing can be carried out at a temperature, pH and oxygen content appropriate for a recombinant cell. Suitable culturing conditions are within the expertise of one of ordinary skill in the art. Examples of suitable conditions are included in the Examples section.
Depending on the vector and host system used for production, resultant proteins of the present invention may either remain within the recombinant cell; be secreted into the fermentation medium; be secreted into a space between two cellular membranes, such as the periplasmic space in E. coli; or be retained on the outer surface of a cell or viral membrane.
The phrase xe2x80x9crecovering the proteinxe2x80x9d, as well as similar phrases, refers to collecting the whole fermentation medium containing the protein and need not imply additional steps of separation or purification. Proteins of the present invention can be purified using a variety of standard protein purification techniques, such as, but not limited to, affinity chromatography, ion exchange chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reverse phase chromatography, concanavalin A chromatography, chromatofocusing and differential solubilization. Proteins of the present invention are preferably retrieved in xe2x80x9csubstantially purexe2x80x9d form. As used herein, xe2x80x9csubstantially purexe2x80x9d refers to a purity that allows for the effective use of the protein as a composition to inhibit T. gondii oocyst shedding in a cat due to infection with T. gondii, or for preventing T. gondii infection in an animal, or as a diagnostic reagent. A composition for inhibiting T. gondii oocyst shedding in a cat due to infection with T. gondii animals, or for preventing T. gondii infection in an animal for example, should exhibit no substantial toxicity and preferably should be capable of stimulating the production of antibodies in a treated animal.
The present invention also includes isolated (i.e., removed from their natural milieu) antibodies that selectively bind to an immunogenic T. gondii protein of the present invention or a mimetope thereof (e.g., anti-T. gondii antibodies). As used herein, the term xe2x80x9cselectively binds toxe2x80x9d an immunogenic T. gondii protein refers to the ability of antibodies of the present invention to preferentially bind to specified proteins and mimetopes thereof of the present invention. Binding can be measured using a variety of methods standard in the art including enzyme immunoassays (e.g., ELISA), immunoblot assays, etc.; see, for example, Sambrook et al., ibid., and Harlow, et al., 1988, Antibodies, a Laboratory Manual, Cold Spring Harbor Labs Press; Harlow et al., ibid., is incorporated by reference herein in its entirety. An anti-T. gondii antibody of the present invention preferably selectively binds to an immunogenic T. gondii protein in such a way as to inhibit the function of that protein.
Isolated antibodies of the present invention can include antibodies in any bodily fluid that has been collected (e.g., recovered) from an animal. Suitable bodily fluids include, but are not limited to, blood, serum, plasma, urine, tears, aqueous humor, central nervous system fluid (CNF), saliva, lymph, nasal secretions, milk and feces. Thus, serum containing antibodies (i.e., antiserum) or mucosal secretions, such as intestinal secretions, are examples of isolated antibodies. Other embodiments of antibodies include antibodies that have been purified to varying degrees. Antibodies of the present invention can be polyclonal or monoclonal, or can be functional equivalents such as antibody fragments and genetically-engineered antibodies, including single chain antibodies or chimeric antibodies that can bind to one or more epitopes.
A preferred method to produce antibodies of the present invention includes (a) administering to an animal an effective amount of a protein, peptide or mimetope thereof of the present invention to produce the antibodies and (b) recovering the antibodies. In another method, antibodies of the present invention are produced recombinantly using techniques as heretofore disclosed to produce T. gondii proteins of the present invention. Antibodies raised against defined proteins or mimetopes can be advantageous because such antibodies are not substantially contaminated with antibodies against other substances that might otherwise cause interference in a diagnostic assay or side effects if used in a composition for inhibiting T. gondii oocyst shedding in a cat due to infection with T. gondii, or for preventing T. gondii infection in an animal.
Antibodies of the present invention have a variety of potential uses that are within the scope of the present invention. For example, such antibodies can be used (a) as compounds to passively immunize a cat in order to inhibit the cat from shedding T. gondii oocysts, (b) as reagents in assays to detect infection by T. gondii and/or (c) as tools to screen expression libraries and/or to recover desired proteins of the present invention from a mixture of proteins and other contaminants.
One embodiment of the present invention includes a method for identifying a nucleic acid molecule encoding an immunogenic T. gondii protein. According to this method, antiserum (comprising either monoclonal or polyclonal antibodies) raised against a T. gondii developmental stage or stages, or against oocysts, is used to immunoscreen a T. gondii genomic expression library or a T. gondii cDNA expression library, and a nucleic acid molecule expressing an immunogenic T. gondii protein is identified by its ability to selectively bind to at least one antibody within the antiserum. As used herein, the term immunoscreen refers to a method in which antibodies are mixed with a sample to determine whether the sample contains a substance to which the antibodies can selectively bind. A substance is identified by its ability to selectively bind to such antibodies. Although general methods to accomplishing immunoscreening of expression libraries are known to those skilled in the art, the exact method to use such a technique to identify T. gondii immunogenic proteins was not previously known. The present invention includes the identification of antisera that are useful in the identification and isolation of nucleic acid molecules encoding T. gondii immunogenic proteins. Such antisera include antiserum raised against T. gondii oocysts, antiserum raised against T. gondii bradyzoites, antiserum raised against T. gondii infected cat gut, and antiserum isolated from a cat immune to T. gondii infection. In one embodiment, antiserum as described above is enriched for antibodies specific to T. gondii gametogenic stages. In a preferred embodiment, polyclonal antiserum is produced by exposing an animal to a T. gondii antigen or antigens, then isolating the antiserum from the animal so exposed. Methods to produce and use the various antisera are described in the Examples section.
In another embodiment, immunoscreening as described above can be used to identify an immunogenic T. gondii protein. According to this method, antiserum as described above is used to immunoscreen a T. gondii genomic expression library or cDNA expression library, and an immunogenic T. gondii protein is identified. T. gondii immunogenic proteins can also be identified by immunoscreening preparations containing T. gondii antigens (e.g., T. gondii oocysts, bradyzoites, infected cat guts) using antiserum as described above.
Nucleic acid molecules and proteins identified using such techniques can be isolated (i.e., recovered) and purified to a desired state of purity using techniques known to those skilled in the art.
One embodiment of the present invention is a composition that, when administered to a cat in an effective manner, is capable of preventing that cat from shedding T. gondii oocysts. Compositions of the present invention, useful for inhibiting T. gondii oocyst shedding in a cat due to infection with T. gondii (i.e., infection with T. gondii causes oocyst shedding in cats), include at least one of the following protective compounds: an isolated immunogenic T. gondii protein or a mimetope thereof, an isolated nucleic acid molecule that hybridizes under stringent hybridization conditions with a nucleic acid molecule comprising one of the nucleic acid molecules and/or nucleic acid sequences cited in Table 1, an isolated antibody that selectively binds to an immunogenic T. gondii protein, an inhibitor of T. gondii function identified by its ability to bind to an immunogenic T. gondii protein and thereby impede development and/or the production of oocysts, or a mixture thereof (i.e., combination of at least two of the compounds). As used herein, a protective compound refers to a compound that, when administered to a cat in an effective manner, is able to inhibit the cat from shedding T. gondii oocysts upon infection with T. gondii. The term protective compound also refers to a compound that, when administered to a cat or other animal, including a human, in an effective manner, is able to prevent or ameliorate disease caused by infection with T. gondii. Examples of proteins, nucleic acid molecules, antibodies and inhibitors of the present invention are disclosed herein.
The present invention also includes a composition comprising at least one T. gondii protein-based compound of the present invention in combination with at least one additional compound protective against one or more infectious agents. Examples of such compounds and infectious agents are disclosed herein.
Compositions of the present invention that are useful for preventing T. gondii infection can be administered to any animal susceptible to such therapy, preferably to mammals.
In order to inhibit a cat from shedding T. gondii oocysts, a composition of the present invention is administered to the cat in a manner effective to inhibit that cat from shedding T. gondii oocysts. In a preferred embodiment, compositions of the present invention are administered to cats prior to infection in order to prevent oocyst shedding (i.e., as a preventative vaccine). In another embodiment, compositions of the present invention can be administered to animals after infection in order to treat disease caused by T. gondii (e.g., as a therapeutic vaccine).
Compositions of the present invention, useful for inhibiting T. gondii oocyst shedding in a cat due to infection with T. gondii, or for preventing T. gondii infection in an animal, can be formulated in an excipient that the animal to be treated can tolerate. Examples of such excipients include water, saline, Ringer""s solution, dextrose solution, Hank""s solution, and other aqueous physiologically balanced salt solutions. Nonaqueous vehicles, such as fixed oils, sesame oil, ethyl oleate, or triglycerides may also be used. Other useful formulations include suspensions containing viscosity enhancing agents, such as sodium carboxymethylcellulose, sorbitol, or dextran. Excipients can also contain minor amounts of additives, such as substances that enhance isotonicity and chemical stability. Examples of buffers include phosphate buffer, bicarbonate buffer and Tris buffer, while examples of preservatives include thimerosal, -or o-cresol, formalin and benzyl alcohol. Standard formulations can either be liquid injectables or solids which can be taken up in a suitable liquid as a suspension or solution for injection. Thus, in a non-liquid formulation, the excipient can comprise dextrose, human serum albumin, preservatives, etc., to which sterile water or saline can be added prior to administration.
In one embodiment of the present invention, a composition useful for inhibiting oocyst shedding in a cat infected with T. gondii, or for preventing T. gondii infection in an animal, can include an adjuvant. Adjuvants are agents that are capable of enhancing the immune response of an animal to a specific antigen. Suitable adjuvants include, but are not limited to, cytokines, chemokines, and compounds that induce the production of cytokines and chemokines (e.g., granulocyte macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), macrophage colony stimulating factor (M-CSF), colony stimulating factor (CSF), erythropoietin (EPO), interleukin 2 (IL-2), interleukin-3 (IL-3), interleukin 4 (IL-4), interleukin 5 (IL-5), interleukin 6 (IL-6), interleukin 7 (IL-7), interleukin 8 (IL-8), interleukin 10 (IL-10), interleukin 12 (IL-12), interferon gamma, interferon gamma inducing factor I (IGIF), transforming growth factor beta, RANTES (regulated upon activation, normal T-cell expressed and presumably secreted), macrophage inflammatory proteins (e.g., MIP-1 alpha and MIP-1 beta), and Leishmania elongation initiating factor (LEIF)); bacterial components (e.g., endotoxins, in particular superantigens, exotoxins and cell wall components); aluminum-based salts; calcium-based salts; silica; polynucleotides; toxoids; serum proteins, viral coat proteins; block copolymer adjuvants (e.g., Hunter""s Titermax(trademark) adjuvant (Vaxcel(trademark), Inc. Norcross, Ga.), Ribi adjuvants (Ribi ImmunoChem Research, Inc., Hamilton, Mont.); and saponins and their derivatives (e.g., Quil A (Superfos Biosector A/S, Denmark). Protein adjuvants of the present invention can be delivered in the form of the protein themselves or of nucleic acid molecules encoding such proteins using the methods described herein.
In one embodiment of the present invention, a composition useful for inhibiting oocyst shedding in a cat infected with T. gondii, or for preventing T. gondii infection in an animal, can include a carrier. Carriers include compounds that increase the half-life of a composition of the present invention in the treated animal. Suitable carriers include, but are not limited to, polymeric controlled release vehicles, biodegradable implants, liposomes, bacteria, viruses, other cells, oils, esters, and glycols.
One embodiment of the present invention is a controlled release formulation that is capable of slowly releasing a composition of the present invention into an animal. As used herein, a controlled release formulation comprises a composition of the present invention in a controlled release vehicle. Suitable controlled release vehicles include, but are not limited to, biocompatible polymers, other polymeric matrices, capsules, microcapsules, microparticles, bolus preparations, osmotic pumps, diffusion devices, liposomes, lipospheres, and transdermal delivery systems. Other controlled release formulations of the present invention include liquids that, upon administration to an animal, form a solid or a gel in situ. Preferred controlled release formulations are biodegradable (i.e., bioerodible).
A preferred controlled release formulation of the present invention is capable of releasing a composition of the present invention into the blood of the treated animal at a constant rate sufficient to attain dose levels of the composition effective to either inhibit oocyst shedding by cats, or to protect an animal from disease caused by T. gondii. The composition is preferably released over a period of time ranging from about 1 to about 12 months. A controlled release formulation of the present invention is capable of effecting a treatment preferably for at least about 1 month, more preferably for at least about 3 months, even more preferably for at least about 6 months, even more preferably for at least about 9 months, and even more preferably for at least about 12 months.
Compositions of the present invention can be administered to cats prior to infection in order to inhibit oocyst shedding, and/or can be administered to cats or other animals, including humans, before infection in order to prevent disease caused by T. gondii infection, or after infection in order to treat disease caused by T. gondii. For example, nucleic acid molecules, proteins, mimetopes thereof, antibodies thereof, and inhibitors thereof can be used to treat or prevent disease caused by T. gondii infection. Acceptable protocols to administer compositions of the present invention include individual dose size, number of doses, frequency of dose administration, and mode of administration. Determination of such protocols can be accomplished by those skilled in the art. A suitable single dose is a dose that is capable of protecting an animal from disease when administered one or more times over a suitable time period. For example, a preferred single dose of a protein, mimetope or antibody composition of the present invention is from about 1 microgram (xcexcg) to about 10 milligrams (mg) of the composition per kilogram body weight of the animal. Booster doses can be administered from about 2 weeks to several years after the original administration. Booster administrations preferably are administered when the immune response of the animal becomes insufficient to protect the animal from disease. A preferred administration schedule is one in which from about 10 xcexcg to about 1 mg of the composition per kg body weight of the animal is administered from about one to about two times over a time period of from about 2 weeks to about 12 months. Modes of administration can include, but are not limited to, injection, oral administration, inhalation, nasal administration, intraocular administration, anal administration, topical administration, particle bombardment, and intradermal scarification. Preferred injection methods include intradermal, intramuscular, subcutaneous, intravenous methods, with intradermal injection and intramuscular injection being more preferred. A particularly preferred method is mucosal administration.
According to one embodiment, a nucleic acid molecule of the present invention can be administered to an animal in a fashion to enable expression of that nucleic acid molecule into a protective protein or protective RNA (e.g., antisense RNA, ribozyme, triple helix forms or RNA drug) in the animal. Nucleic acid molecules can be delivered to an animal in a variety of methods including, but not limited to, (a) administering a nucleic acid not packaged in a viral coat or cell as a genetic vaccine (e.g., as xe2x80x9cnakedxe2x80x9d DNA or RNA molecules with or without a non-viral/non-cellular carrier (e.g., liposome, hydrogel, etc.) or (b) administering a nucleic acid molecule packaged as a recombinant virus vaccine or as a recombinant cell vaccine (i.e., the nucleic acid molecule is delivered by a viral or cellular vehicle).
A genetic vaccine of the present invention includes a recombinant molecule of the present invention. As such, a genetic vaccine comprises at least one isolated nucleic acid molecule encoding an immunogenic T. gondii protein operatively linked to a eukaryotic or prokaryotic transcription control region. A genetic vaccine can be either RNA or DNA, can have components from prokaryotic as well as eukaryotic sources, and can have the ability, by methods described herein, to enter either eukaryotic or prokaryotic cells and direct expression of isolated nucleic acid molecules of the present invention in those cells. In a preferred embodiment, a genetic vaccine of the present invention includes a recombinant virus genome (i.e., a nucleic acid molecule of the present invention ligated to at least one viral genome in which transcription of the nucleic acid molecule is directed either by a transcription control region on the genome or a separate transcription control region) or a recombinant plasmid that includes a nucleic acid molecule of the present invention ligated into a vector that is not a viral genome such that the nucleic acid molecule is operatively linked to a transcription control region.
A genetic vaccine of the present invention includes a nucleic acid molecule of the present invention and preferably includes a recombinant molecule of the present invention that preferably is replication, or otherwise amplification, competent. A genetic vaccine of the present invention can comprise one or more nucleic acid molecules of the present invention in the form of, for example, a dicistronic recombinant molecule. Preferred genetic vaccines include at least a portion of a viral genome (i.e., a viral vector) and a nucleic acid molecule of the present invention. Preferred viral vectors include those based on alphaviruses, poxviruses, adenoviruses, adeno-associated viruses, herpesviruses, picomaviruses, and retroviruses, with those based on alphaviruses (e.g., Sindbis virus or Semliki forest virus), picornaviruses (e.g., poliovirus or mengovirus), species-specific herpesviruses and poxviruses being particularly preferred. Any suitable transcription control sequence can be used, including those disclosed as suitable for protein production. Particularly preferred transcription control sequences include cytomegalovirus immediate early (preferably in conjunction with Intron-A), Rous sarcoma virus long terminal repeat, and tissue-specific transcription control sequences, as well as transcription control sequences endogenous to viral vectors if viral vectors are used. The incorporation of a xe2x80x9cstrongxe2x80x9d polyadenylation signal is also preferred.
Genetic vaccines of the present invention can be administered in a variety of ways, with intramuscular, subcutaneous, intradermal, transdermal, intraocular, intranasal and oral routes of administration being preferred. A preferred single dose of a genetic vaccine ranges from about 1 nanogram (ng) to about 600 xcexcg, depending on the route of administration and/or method of delivery, as can be determined by those skilled in the art. Suitable delivery methods include, for example, by injection, by gene gun, as drops, as inhaled aerosols, ingested in microparticles or microcapsules, and/or topical delivery. Genetic vaccines of the present invention can be contained in an aqueous excipient (e.g., phosphate buffered saline) alone or in a carrier (e.g., lipid-based vehicles).
A recombinant virus vaccine of the present invention includes a recombinant molecule of the present invention that is packaged in a viral coat and that can be expressed in an animal after administration. Preferably, the recombinant molecule is packagingxe2x80x94or replicationxe2x80x94deficient and/or encodes an attenuated virus. A number of recombinant viruses can be used, including, but not limited to, those based on alphaviruses, poxviruses, adenoviruses, herpesviruses, picomaviruses, and retroviruses. Preferred recombinant virus vaccines are those based on alphaviruses (e.g., Sindbis virus), picomaviruses (e.g., poliovirus, mengovirus), raccoon poxviruses, species-specific herpesviruses and species-specific poxviruses. An example of methods to produce and use alphavirus recombinant virus vaccines are disclosed in PCT Publication No. WO 94/17813, by Xiong et al., published Aug. 18, 1994, which is incorporated by reference herein in its entirety.
When administered to an animal, a recombinant virus vaccine of the present invention infects cells within the immunized animal and directs the production of a protective protein or RNA nucleic acid molecule that is capable of preventing a cat from shedding oocysts as disclosed herein. For example, a recombinant virus vaccine comprising a nucleic acid molecule encoding an immunogenic T. gondii protein of the present invention is administered according to a protocol that results in the subject cat producing a sufficient immune response to inhibit shedding T. gondii oocysts. A preferred single dose of a recombinant virus vaccine of the present invention is from about 1xc3x97104 to about 1xc3x97108 virus plaque forming units (pfu) per kilogram body weight of the animal. Administration protocols are similar to those described herein for protein-based vaccines, with subcutaneous, intramuscular, intraocular, intranasal and oral administration routes being preferred.
A recombinant cell vaccine of the present invention includes recombinant cells of the present invention that express at least one protein of the present invention. Preferred recombinant cells for this embodiment include Salmonella, E. coli, Listeria, Mycobacterium, S. frugiperda, yeast, (including Saccharomyces cerevisiae and Pichia pastoris), BHK, CV-1, myoblast G8, COS (e.g., COS-7), Vero, MDCK and CRFK recombinant cells. Recombinant cell vaccines of the present invention can be administered in a variety of ways but have the advantage that they can be administered orally, preferably at doses ranging from about 108 to about 1012 cells per kilogram body weight. Administration protocols are similar to those described herein for protein-based vaccines. Recombinant cell vaccines can comprise whole cells, cells stripped of cell walls or cell lysates.
The efficacy of a composition of the present invention to inhibit oocyst shedding caused by T. gondii can be tested in a variety of ways including, but not limited to, detection of protective antibodies (using, for example, proteins or mimetopes of the present invention), detection of cellular immunity within the treated animal, or challenge of the treated animal with T. gondii to determine whether the treated animal is resistant to oocyst shedding. Challenge studies can include direct administration of T. gondii tachyzoites or tissue cysts or sporulated oocysts (the infective stages) to the treated animal. In one embodiment, compositions of the present invention can be tested in animal models such as mice. Such techniques are known to those skilled in the art.
One preferred embodiment of the present invention is the use of immunogenic T. gondii proteins, nucleic acid molecules encoding immunogenic T. gondii proteins, antibodies and inhibitors of the present invention, to inhibit a cat from shedding oocysts. It is particularly preferred to prevent intestinal stages of the parasite from developing into oocysts. Preferred compositions are those that are able to inhibit at least one step in the portion of the parasite""s development cycle that occurs in the intestines prior to the development of oocysts. In cats infected with tissue cysts, for example, the prepatent period for oocyst shedding is three to five days. When cats are infected with sporulated oocysts, for example, the prepatent period can range from 19 to 45 days. Particularly preferred compositions useful for inhibiting oocyst shedding in a cat infected with T. gondii include T. gondii-based compositions of the present invention. Such compositions include nucleic acid molecules encoding immunogenic T. gondii proteins, immunogenic T. gondii proteins and mimetopes thereof and anti-T. gondii antibodies. Compositions of the present invention are administered to cats in a manner effective to inhibit the cats from shedding T. gondii oocysts. Additional protection may be obtained by administering additional protective compounds, including other T. gondii proteins, nucleic acid molecules and antibodies, as disclosed herein.
It is also within the scope of the present invention to use isolated proteins, mimetopes, nucleic acid molecules and antibodies of the present invention as diagnostic reagents to detect infection by T. gondii. These diagnostic reagents can further be supplemented with additional compounds that can specifically detect any or all phases of the parasite""s life cycle. General methods to use diagnostic reagents in the diagnosis of disease are known to those skilled in the art. A method or a kit for the detection of T. gondii infection could be combined with reagents for the detection of additional infectious agents, for example viruses (e.g. Coronaviruses), bacteria (e.g. Campylobacter, Clostridium, Salmonella), protozoa (e.g. Cryptosporidium, Giardia, Isospora, Hammondia, Sarcocystis, Besnoitia, Microsporidium), and/or multi-cellular organisms (e.g. Teania, Anclostoma, Toxocara, Physaloptera, Paragonimus, Strongyloides, Trichuris).
Another embodiment of the present invention is a method to detect microscopic parasite cysts or oocysts in feces using PCR amplification techniques. By microscopic, it is meant cysts or oocysts that are too small to be conveniently detected by simple visual observation of the feces. Preferred organisms to be detected include oocysts from infectious protozoan parasites including members of the apicomplexa and others including, for example, Toxoplasma, Cryptosporidium, Isospora, Giardia, Eimeria, Hammondia, Sarcocystis, Besnoitia, Microsporidium. Additional infectious agents to detect include, for example, viruses (e.g. Coronaviruses), bacteria (e.g. Campylobacter, Clostridium, Salmonella), and/or multi-cellular organisms (e.g. Teania, Anclostoma, Toxocara, Physaloptera, Paragonimus, Strongyloides, Trichuris). Particularly preferred oocysts to be detected include Toxoplasma and Cryptosporidium oocysts. Preferred cysts to be detected include any cysts capable of binding to a solid support and remaining bound to the support through a washing step. Preferred cysts include Giardia cysts. According to this embodiment of the invention, a solid support that is capable of binding cysts or oocysts is contacted with a sample of feces, which may or may not have been partially solubilized first in an aqueous solution, and the sample of feces is allowed to dry on the support. The solid support can be of any material to which the cysts or oocyts will bind and remain bound during washing in an aqueous solution. The support can comprise one or more compounds that aid in PCR amplification of the sample, for example by allowing the inhibitors to be released in the wash step, or by binding inhibitors of PCR that are not released in the elution step, or by otherwise inactivating inhibitors of PCR amplification. Preferred supports comprise a paper substrate to which the oocysts or cysts can bind. Preferred supports include IsoCodeJ(trademark) Stix, or their equivalent, SandS(copyright) #903(trademark), or their equivalent, or Nobuto Blood Filter Strips, or their equivalent. The support, or the portion of the support contacted with the sample of feces, is preferably small enough to fit into a container convenient for the wash step; eg., a size that will fit into a 1.5. ml conical centrifuge tube. The portion of the support that is contacted with the sample of feces can be removed from the rest of the support in order to achieve a convenient size. The portion of the support that includes the dried sample of feces is then washed with an aqueous solution. In a preferred embodiment the aqueous solution is water, preferably distilled water. The solution can comprise one or more compounds that aid in PCR amplification of the sample, for example by inactivating or removing inhibitors of PCR amplification. DNA associated with the sample is eluted by adding an aqueous solution to the support and then heating the solution to a temperature sufficient to elute DNA from the sample, into the solution. In a preferred embodiment, the aqueous solution into which the sample is eluted is water, preferably distilled water. This solution can comprise one or more compounds that aid in PCR amplification of the sample, for example by inactivating inhibitors of PCR amplification, or by improving reaction conditions for the PCR reaction. The heating step comprises heating to a temperature sufficient to elute DNA from the sample. A preferred temperature is approximately 95xc2x0 C. Oocyst or cyst-specific DNA in the elution solution is then PCR amplified using primers specific to the oocysts or cysts being detected. The amplification products indicative of oocysts or cysts are then detected using any means available for the detection of PCR amplification products. These can include, for example, separation and observation of the PCR products on a gel, or detection and/or quantification by PCR ELISA. In a preferred embodiment of the present invention, nucleic acid molecules of the present invention are used for the detection of T. gondii oocysts in cat feces by PCR amplification using nucleic acid molecules of the present invention as primers. According to the present invention, detection of oocysts can be accomplished by direct analysis of feces. Methods to conduct such an assay are described further in the Examples section.
The following examples are provided for the purposes of illustration and are not intended to limit the scope of the present invention.